What Does Physiological Mean?

S © t a r ecentl y r e vie wed an original paper with a title including he words “physiological stimulation.” In this particular case, t turned out that the stim ulation w as far fr om physiological. he concentration of the hormone used for acti v ation w as w ay bov e the maximal level ever observed in vivo, and this made e think about the use, and misuse, of the word “physiologial.” It is a word that we (physiologists) employ fr equentl y and erhaps too fr equentl y. Papers in physiological, and other, jourals often refer to “physiological conditions,” which sometimes s taken to indicate experiments in vi v o, but also fr equentl y ust means that experiments on single cells or tissue fragents were carried out with stimulation protocols and under ircumstances that are not unlike those that could happen n vi v o. We hav e li v ed thr ough a long and pr oducti v e period of singleell biology. Very important discoveries of real significance have een made, but it is now becoming incr easingl y clear that ther e r e many criticall y important inter actions betw een different djacent cell types in most tissues. To c har acterize these proesses, it is necessary to observe simultaneously more than one ell type in individual organs or tissues. Furthermore, the behavor of a particular cell type in isolation may not be the same as hen it is embedded in its normal environment. Of particular oncern is the tacit assumption in many studies that processes n cell lines reflect those in normal cells in situ. It may therefore e useful to reflect on the usefulness of working under real physological conditions, notwithstanding the obvious difficulties of oing so. In what follows, I’ll try to illuminate these issues by xamples from immunology, epithelial physiology, and neurocience. Ca 2 + signaling studies in imm une cells hav e been immensel y uccessful in unravelling key Ca 2 + transport events and, in articular, the properties of the Ca 2 + r elease acti v ated Ca 2 + CRAC) channel of the Orai type and its molecular control echanism. 1 Unlike the situation in epithelial cells, where Ca 2 +

I r ecentl y r e vie wed an original paper with a title including the words "physiological stimulation." In this particular case, it turned out that the stim ulation w as far fr om physiological. The concentration of the hormone used for acti v ation w as w ay a bov e the maximal level ever observed in vivo, and this made me think about the use, and misuse, of the word "physiological." It is a word that we (physiologists) employ fr equentl y and perhaps too fr equentl y. Papers in physiological, and other, journals often refer to "physiological conditions," which sometimes is taken to indicate experiments in vi v o, but also fr equentl y just means that experiments on single cells or tissue fragments were carried out with stimulation protocols and under circumstances that are not unlike those that could happen in vi v o.
We hav e li v ed thr ough a long and pr oducti v e period of singlecell biology. Very important discoveries of real significance have been made, but it is now becoming incr easingl y clear that ther e ar e many criticall y important inter actions betw een different adjacent cell types in most tissues. To c har acterize these processes, it is necessary to observe simultaneously more than one cell type in individual organs or tissues. Furthermore, the behavior of a particular cell type in isolation may not be the same as when it is embedded in its normal environment. Of particular concern is the tacit assumption in many studies that processes in cell lines reflect those in normal cells in situ. It may therefore be useful to reflect on the usefulness of working under real physiological conditions, notwithstanding the obvious difficulties of doing so. In what follows, I'll try to illuminate these issues by examples from immunology, epithelial physiology, and neuroscience.
Ca 2 + signaling studies in imm une cells hav e been immensel y successful in unravelling key Ca 2 + transport events and, in particular, the properties of the Ca 2 + r elease acti v ated Ca 2 + (CRAC) channel of the Orai type and its molecular control mechanism. 1 Unlike the situation in epithelial cells, where Ca 2 + signaling studies have largely been conducted on normal cells, 2 v er y many studies on immune cells have been conducted on cell lines and have focused on defining molecular pathways rather than describing physiological signaling patterns. The studies of Markus Hoth and colla borators hav e , how ev er, r ev ealed important aspects of physiological Ca 2 + signaling. 3 , 4 One critical element of the physiology of T cell acti v ation is the role of the immunological synapse. 4 This is a specialized junction between a T cell and an antigen-presenting cell that esta b lishes efficient communication between the cells, thereby promoting activation of the immune response. The generation of the immunological synapse focuses the Ca 2 + signaling domain at the inner mouth of the CRAC/Orai1 channel by translocating the mitochondria close to interaction points between the CRAC channels and the endoplasmic reticulum Ca 2 + sensor STIM1. The critical role of the immunological synapse dictates that the T cell becomes functionally polarized and, like epithelial cells, 2 can generate both local and global Ca 2 + signals. 3 , 4 The immunological synapse allows the creation of large Ca 2 + signals locally, immediately below the plasma membr ane , in such a way that the hotspot of Ca 2 + entry occurs at some distance from the site of the Ca 2 + pumps in the plasma membr ane , pr ev enting immediate expulsion of Ca 2 + entering through the CRAC channels. 4 Ca 2 + signaling has r ecentl y been studied in normal macr opha ges embedded in the exocrine pancreatic tissue. 5 These endogenous immune cells display oscillatory Ca 2 + signals in response to activation of various purinergic receptors, but do not normally respond to stimulation with IgG . Ho wever, after induction of alcohol-r elated pancr eatitis, when there is major recruitment of macrophages into the pancreas, these cells generate r e petiti v e Ca 2 + spikes when challenged in this w ay. 5 In pancr eatitis, many pr oducts, including ADP and ATP from dying acinar cells, appear in the immediate environment of the endogenous macr opha ges, acti v ating these cells.

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In epithelia, and particularly exocrine gland epithelia, there has been a long tradition for Ca 2 + signaling studies in normal cells. 2 Such studies gave rise to the concept of Ca 2 + signal generation by primary release of Ca 2 + from intracellular stores 2 , 5 and later to the identification of IP 3 as an intracellular messenger. 2 Furthermore, the important concept of physiological local Ca 2 + signaling and pathological global Ca 2 + signaling arose from single-cell studies and was later verified by work on lobule pr e par ations in whic h the normal microscopic structure of the gland was preserved. 2 Recently , T akahiro T akano and David Yule have carried these studies further by examining Ca 2 + signal generation in pancreatic acinar cells in the intact pancreas in the living mouse. 6 This in vivo study showed that physiological activation, either by electrical stimulation of the vagal nerve, infusion of cholecystokinin (CCK) or intake of a meal, elicited Ca 2 + signals in the acinar cells. At low levels of stimulation, these signals were predominantly local, confined to the apical granulecontaining r egion, wher eas at higher intensities of stim ulation, the signals became globalized. 6 Thus, this new in vi v o study confirmed, in a real physiological setting, earlier work on isolated cells and pancreatic lobules. 2 An important new and unexpected finding from the in vivo study was the appearance of low level Ca 2 + signaling events, even in the complete absence of external stimulation, in a sub-population of acinar cells. This was due to the endogenous basal CCK level in the blood, since it was abolished by a CCK antagonist, but not by atropine. 6 The inte gr ati v e neur oscientists hav e fr equentl y been mor e ad vanced tec hnically than other physiologists and have perhaps also been particularly concerned about studying physiologicall y r elev ant situations. Thus, alr eady mor e than 15 yr a go, Carl Petersen and his group showed that the cortical r e pr esentation of the physiologically important whisker touch could be studied by v olta ge-sensiti v e dye ima ging in fr eel y moving mice. 7 Technicall y, this pr ov ed the usefulness of fiber optics to image cortical sensor y acti vity with high r esolution and, importantl y, the results of these studies demonstrated differential processing of sensory input depending on behavior. 7 , 8 In a v er y r ecent study of neurons and astrocytes in awake and behaving mice, Alex Verkhratsky and collaborators have shown that locomotion induces fundamentally different patterns of Ca 2 + signaling in astrocytes and neurons. 9 Whereas the neuronal Ca 2 + concentration increased almost immediately after the onset of locomotion and faithfully followed such e pisodes, the astr ocytic Ca 2 + signals wer e dela yed b y se veral seconds, developing on a different timescale. 9 These findings provide important fresh evidence for the special and distinct roles pla yed b y the astr ocytes in sustaining neur ons and supporting their function. 9 , 10 These are just a few examples of findings that could only have been made by studies paying close attention to the establishment of real physiological conditions. When I started out in resear c h, more than 50 yr ago, we generally did work in vi v o under proper physiological conditions, but we did not have tools to get mechanistic information about cellular control mechanisms. This became possible in single-cell studies carried out in the 1980s, 1990s, and 2000s. Now, we have tools that allow us to observe molecular events simultaneously in multiple cell types in intact tissues and incr easingl y in li ving behaving animals. In the future , w e may perhaps be a b le to do some of these studies safely in humans and might then also carry out investigations under critical pathological conditions. This would open up amazing new vistas of great consequence for diagnosis and treatment.

Funding
None declared.

Conflict of Interest
O.H.P. holds the position of Editor-in-Chief for FUNCTION and is b linded fr om r e vie wing or making decisions for the manuscript.

Da ta Av ailability
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